Seeking compounds preferentially potent and selective for MMP-13, we reported in the preceding Letter on a series of hydroxamic acids with a flexible benzamide tail groups.(1a) Here, we replace the amide moiety with non-hydrolyzable heterocycles in an effort to improve half-life. We identify a hydroxamate tetrazole 4e that spares MMP-1 and -14, shows \u3e400-fold selectivity versus MMP-8 and \u3e600-fold selectivity versus MMP-2, and has a 4.8 h half-life in rats. X-ray data (1.9 Å) for tetrazole 4c is presented

Barta, Thomas E.

Becker, Daniel P.

Bedell, Louis J.

Easton, Alan M.

Name not available

MMP-13 Selective α-sulfone Hydroxamates: a Survey of P1\u27 Heterocyclic Amide Isosteres

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Loyola University ChicagoLoyola eCommonsChemistry: Faculty Publications and Other Works Faculty Publications5-15-2011MMP-13 Selective α-sulfone Hydroxamates: aSurvey of P1' Heterocyclic Amide IsosteresThomas E. BartaPfizer Research & DevelopmentDaniel P. BeckerLoyola University Chicago, dbecke3@luc.eduLouis J. BedellPfizer Research & DevelopmentAlan M. EastonLoyola University ChicagoAuthor ManuscriptThis is a pre-publication author manuscript of the final, published article.This Article is brought to you for free and open access by the Faculty Publications at Loyola eCommons. It has been accepted for inclusion inChemistry: Faculty Publications and Other Works by an authorized administrator of Loyola eCommons. For more information, please contactecommons@luc.edu.This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.© 2011 ElsevierRecommended CitationBarta, Thomas E.; Becker, Daniel P.; Bedell, Louis J.; and Easton, Alan M.. MMP-13 Selective α-sulfone Hydroxamates: a Survey of P1'Heterocyclic Amide Isosteres. Bioorganic & Medicinal Chemistry Letters, 21, 10: , 2011. Retrieved from Loyola eCommons,Chemistry: Faculty Publications and Other Works, http://dx.doi.org/10.1016/j.bmcl.2011.03.099A Survey of Isosteric Amide Replacements in a Series of Extended, Hydroxamate-containing, Selective MMP-13 InhibitorsThomas E. Barta, Daniel P. Becker, Louis J. Bedell, Alan M. Easton, Susan L. Hockerman, James Kiefer, Grace E. Munie, Karl J. Mathis, Madeleine H. Li, Joseph G. Rico, Clara I. Villamil,  Jennifer M. WilliamsAbstract: Seeking compounds preferentially potent and selective for MMP-13, we recently reported on a series of hydroxamic acids with a flexible benzamide tail groups.1 In this Letter, we replace the amide moiety with non-hydrolyzable heterocycles in an effort to improve half-life. We identify a hydroxamate tetrazole 4e that spares MMP-1 and -14,  shows >400 fold selectivity vs. MMP-8 and >600 fold selectivity vs. MMP-2, and has a 4.8 h half-life in rats.Matrix Metalloproteinases (MMPs) are a family of about 27 zinc-dependent enzymes responsible for the turnover of collagen in connective tissue. There are a variety of disease states where degradation of collagen contributes to the pathology, specifically, in osteo- and rheumatoid arthritis; in tumor angiogenesis and metastasis; in post-MI cardiovascular remodeling. In the previous Letter,1a we note that joint-stiffening, "musculoskeletal syndrome" (MSS), upon prolonged dosing with MMP inhibitors (MMPi’s) has prevented  approval of an MMPi. Efforts have been made to spare MMP isoforms that are thought to contribute to MSS, specifically, MMP-1 and -14 (MT1-MMP) (SD-2590, fig. 1),1b but a more focused approach toward realizing efficacious MMP inhibitors with reduced side effects should be to focus on optimizing a single MMP isoform that confers the most therapeutic benefit, reducing the probability of off-target protease binding. MMP-13, upregulated in osteoarthritic joints and in cancer, is an attractive isoform to target, and structural studies show that -13 differs from other MMP's deep in the S1' pocket, suggesting that lengthier inhibitors may confer selectivity.With this in mind, we elaborated a series of rigid piperidino- ketones (compound 2, Figure 1) with  lengthier P1' subunits and, with optimization, we were able to achieve significant selectivity for MMP-13 vs. other MMP isoforms.2 Conceptually dissecting the piperidine ring in compound 2, we also wanted to explore acyclic-chain analogs such as amide 3 (Figure 1).1While excellent MMP-13 selectivity (Table 1 ) was achieved with a number of amides related to compound 3, the amides displayed short half-life in rats, presumably due to hydrolytic cleavage of the amide bond. In an effort to prepare compounds with suitable selectivity and PK, we incorporated the SAR findings from the acyclic amide series into a small series of compounds where the amide is replaced by isosteric heterocycles. This Letter summarizes that effort.Among the isosteres we wanted to consider were tetrazoles. Entry into this series began by alkylating the requisite 5-phenyl-1H-tetrazoles (e.g. 5, scheme 1) using O-tetrahydropyranyl-3-chloropropanol. Alkylation afforded predominantly the desired 2-isomers for 5-[4-methoxyphenyl]- and 5-[4-trifluoromethoxyphenyl]-tetrazole, and the products were purified by chromatography. The THP protecting group could be removed with acid and the product alcohol (e.g. 6) reacted with the known aryl fluoride 7.1 The t-butyl ester 8 was cleaved and the resulting acid reacted with O-tetrahydropyranyl hydroxylamine in the presence of EDC. Removal of the THP gave the desired hydroxamic acids 4b. In the case of 5-[4-methyl-2-pyridyl]-tetrazole, alkylation gave a 3:2 mixture, still favoring the 2-position. The poor selectivity in the alkylation step afforded us the opportunity to isolate meaningful quantities of both 2- and 1- isomers and to carry the isomers individually on to final product (4d and 4e, table 1).Oxadiazoles (e.g 4f, Table 1) were prepared by combining the known acid 91 and 4-(trifluoromethoxy)benzamidoxime in the presence of EDC, then heating the coupled product to close the ring. After condensation of the oxadiazole, the t-butyl ester (10, Scheme 2) is carried on to the hydroxamic acid employing the same transformations used for the tetrazoles.From Table 1, we see that replacing the amide in 3 (Table 1) with various aryl heterocycle combinations: tetrazoles (4a-e); oxadiazoles (4f,g); and imidazoles (4h,i) continues to  demonstrate high levels of selectivity for MMP-13 vs. MMP-1, -2, -7, -8, -9, and -14. Selectivity vs. MMP-3 is lowered. Unexpectedly, we see that the 1,5-isomer of the pyridyl analog (4e) has a selectivity profile very similar to its 2,5-substituted isomer (4d). These results inspired us to make some similarly disposed imidazoles (4j). We also made an isomeric 1,5-substituted tetrazole, where the tetrazole carbon connects to the acyclic chain, by a chemospecific method5 (scheme 2) not involving N-alkylation (4j).The crystal structure of tetrazole 4c was determined at 1.9Å resolution (Figure 2). 6 The selectivity in this series derives from the depth and flexibility of the MMP-13 S1’ pocket, which is distinct from other MMP family members such as MMP-1 and MMP-2, with shorter pockets. The tetrazole group forms a single direct hydrogen bond to the protein backbone at Thr245 (3.0Å); the remainder of the side chain interactions with the protein are van der Waals contacts. The S1’ pocket of MMP-13 readily morphs to interact with inhibitors of various sizes and shapes, as we see in the case of tetrazoles 4d and 4e. We were able to achieve >103 selectivity vs. most other MMP family members with 4c, but even more specific compounds may be possible with this or other scaffolds.We were able to improve on the 0.59 h half-life we measured for the lead amide compound 3; from Table 1, we see heterocyclic compounds with half-lifes ranging from 1.4 to 4.8 h, some compounds having good BA (e.g. tetrazole 4c, 26%; and oxadiazole 4f, 28%) and achieving encouraging Cmax. Based on our data, we believe selected heterocyclic analogs in this Letter would be promising drug candidates for the treatment of arthritis, cancer, and post-MI left ventricular hypertrophy (CHF).References and Notes1. (a) Barta, T. E.; Becker, D.P.; Freskos, J. N.; Fobian, Y.; Heintz, R.; Kiefer, J. R.; Mischke, B. V.; Mullins, P. Preceding paper, this Journal. b) Becker, D. P.; Barta, T. E. Bedell, L. J.; Boehm, T. L. Bond, B. R.; Carroll, J.; Carron, C. P.; DeCrescenzo, G. A.; Easton, A. M.; Freskos, J. N.; Funckes-Shippy, C. L.; Heron, M.; Hockerman, S. L.; Howard, S. C.; Kiefer, J. R.; Li, M. H.; Mathis, K. J.; McDonald, J. J.; Mehta, P. P.; Munie, G. E.; Sunyer, T.; Swearingen, C. A.; Villamil, C. I.; Welsch, D.; Williams, J. M.; Yu, Y.; Yao, J. J. Med Chem. in press.2. Kolodziej, S. A.; Hockerman, S. L.; DeCrescenzo, G. A.; McDonald, J. J.; Mischke, D. A.; Munie, G. E.; Fletcher, T. R.; Stehle, N.; Swearingen, C.; Becker, D. P. Bioorg. and Med. Chem. Lett. 2010, 20, 3561. See also: Kolodziej, S. A.; Hockerman, S. L.; Boehm, T. L.; DeCrescenzo, G. A.; McDonald, J. J.; Mischke, D. A.; Munie, G. E.; Fletcher, T. R.; Stehle, N.; Swearingen, C.; Becker, D. P. Bioorg. and Med. Chem. Lett. 2010, 20, 3557.3. For a conceptually related series, see: Wu, J.; Rush III, T.S.; Hotchandani, R.; Du, X.; Geck, M.; Collins, E.; Xu, Z.B.; Skotnicki, J.; Levin, J.I.; Lovering, F.E. Bioorg. and Med. Chem. Lett. 2005, 15, 4105-4109.4. Komamura, T; Tawara, G.; Yoshihiko, S. JP Patent 63301035 A, 1988; Chem Abstr. 1989, 111, 105859.5. De Lombaert, S.; Blanchard, L.; Stamford, L.B.; Tan, J.; Wallace, E.M.; Satoh, Y.; Fitt, J.; Hoyer, D.; Simonsbergen, D.; Moliterni, J.; Marcopoulos, N.; Savage, P.; Chou, M.; Trapani, A.J.; Jeng, A.Y. J. Med. Chem. 2000, 43, 488-504.6. Coordinates deposited in the protein data bank. Crystallographic conditions detailed in the Supplementary Material.OSNHOHOO OOOSNHOHOO OO NHOOMeSNHOHOO ONN OOMeNNNNOMeNSNHOHOO OO123O4aFigure 1. MMP inhibitors. Compound 1 (SD-2590) spares MMP-1; Compounds 2, 3, and 4a are MMP-13 selective.OCF3O SD-2590NSNHOHOO OO NN NNOCF3NSOO O OO NNNNOCF3NSOO O OFHNNNNOCF3+NNNNOCF3HOcdefScheme 1. Reagents and Conditions:  (a) NaH (2.1 eq), NMP, rt; then 2-(3-chloropropoxy)tetrahydro-2H-pyran (1.1 eq) r.t. to 70°C (45%) (b) AcCl (3.8 eq), MeOH, rt (94%) (c) 6 (1.05 eq), NaH (1.1 eq), NMP, 55°C, 1h (70%) (d) NaOH (50% aq, 10 eq), THF, EtOH, rt to 60°C, then acidify w/ HCl, conc., azeotrope CH3CN (e) triethylamine (XS), HOBT, THPONH2 (1.5 eq based on 7a), EDC (1.5), DMF, 40 h (65%) (f) AcCl (4 eq), MeOH, trit. ether (74%)56ab7a84bOOOOSOO O OOOCF3OSOO O OOONHNNNNOCF3OSOO O OOOOHabScheme 2. Reagants and Conditions: (a) DMF, HOBT (1.4 eq), triethylamine (1.2 eq), 4-(trifluoromethoxy)benzamideoxime (1.2 eq), EDC (1.5 eq), rt, 2h (b) toluene, 90°C, 30 h (c) HOBT (1.33 eq), 4-(trifluromethoxy)aniline (1.33 eq), EDC (1.33 eq), DMF, rt, 4 h (80%); (d) PPh3 (2.0 eq), dioxane; then DEAD (2.0 eq); then TMS-N3 (2.0 eq), rt, 16 h (66%).OSOO O OO NNOOCF3cd910114jXSNHOHOO Ocmpd #b,cO RhMMP inhibition Ki (nM)aNNNNONNNNOCF3NNNNF3CONONOCF3NNNN NNNNNN-2       -3       -7       -8       -9       -13NNF3CONNOCF31300c55048c410230430410920053090c >10kc 1200c 1200c 0.70c4001103.044 300097024025037024000.660.60900 - 900 >10k 1.639c - 190c 1100c 0.20c28 >10k 170 1200 0.66a.) MMP-1 and -14 >10k nM except for 1 (MMP-14 = 14 nM) and 4a (MMP-1 = 5600 nM).b.) For 4a-k, X = O except: 4c, where X = NCH2CH2OMe and 4g, X = NCH2CH3c.) IC50 instead of Ki.31 >10k 180 1600 1.59.9 7100 72 1700 1.28.7 8600 8000 160 0.13130 >10k 140 3.7>10k59 6000 74 1800 1.7Table 1: Selectivity and PK of heterocyclic MMP-13 selective analogs.<0.1c 29c 7000 1.7c 0.2c <0.1cRR1 =A B C D EF G H410 37 >10k 260 370 2.0Rat PKBA%  t1/2 (h)  Cmax ng/mL (MPK)  Cl (mL/min/kg)1 N/AN/A2N/A34a4b4cb4f4gb4d4h4e4j32 2.4ABBCCDEFGH122228 2.41.4 14191.7 2.0 465.0 4.8 631.5 0.59 0.02 (20)61 (1.5)26 1.3 1400 (20)240 (20)2200 (20)3500 (5)3000 (5)7.3 4.0 423500 (5)4i4200 (20)1.21468 2.9 21900 (20)Figure 2.  Structure of MMP-13 with compound 4c bound at the catalytic site. The inhibitor binds deeply within the S1’ pocket of the active site and causes a conformational change opening the loop to accommodate the compound.  The hydroxamate moiety chelates the zinc ion at two positions, and Glu223 forms a hydrogen bond to the hydroxamate.  Similarly, the hydroxamate nitrogen atom forms a hydrogen bond to the protein backbone, as does one of the sulfone oxygen atoms.  A single hydrogen bond is formed between the tetrazole and the protein backbone. A HEPES buffer molecule bound adjacent to the inhibitor in two of the four protein molecules in the crystal, and an array of solvent atoms form an auxiliary van der Waals surface between the inhibitor and the protein.6Supplementary Material:Rod shaped crystals of the MMP-13 in complex with tetrahydro-N-hydroxy-4-[[4-(phenylmethyl)-1-piperazinyl]sulfonyl]-2H-pyran-4-carboxamide, hydrochloride (Getman, Daniel P.; Becker, Daniel P.; Barta, Thomas E.; Villamil, Clara I.; S. L. Hockerman; Bedell, Louis J.; Li, Madelleine H.; Freskos, John N.; Heintz, Robert M.; McDonald Joseph J. ; DeCrescenzo, Gary A. , “Thioaryl Sulfonamide Hydroxamic Acid Compounds” US 6,087,359, 2000.) complex were grown at 4°C by sitting drop vapor diffusion using 5 mg/mL protein and a reservoir solution of 1.4M lithium sulfate, 0.1M Hepes, pH 7.7. The ratio of protein to reservoir solution in the crystallization drop was 5:1.  Crystals were pre-equilibrated in a soaking solution containing 1.5M lithium sulfate, 0.1M Hepes, pH 7.7 without inhibitor at room temperature for more than two hours. These crystals were then transferred to a second solution containing 1.5M lithium sulfate, 0.1M Hepes, pH 7.7 and 2.5mM of replacement inhibitor and were left to incubate at room temperature for several days. The cryo solution consisted of 20% sucrose, 1.5M lithium sulfate, and 0.1M Hepes, pH 7.7.  Data were collected using a Rigaku Micromax 007 X-ray generator on a Mar Research MarCCD 165 detector.  The diffraction data were integrated and scaled with HKL-2000 (Otwinowski, Z. and Minor, W, (1997). In Methods in Enzymology, 276, edited by C.W. Carter, Jr. & R.M. Sweet pp. 307-326, New York:Academic Press.), and the structures were determined by molecular replacement using a prior internal structure of MMP-13 as the initial model.  The model adjustment occurred as in Kjeldgaard (Jones, T.A., Zou, J.Y., Cowan, S.W. and Kjeldgaard, M. Acta Cryst 1991 A47, 110-119.) and Coot (Emsley, P.; Cowtan, K. Acta Crystallographica Section D 2004, 60, 2126), and the structures were refined initially with X-PLOR (Brünger, A. T. 1992. X-plor version 3.1: a system for X ray crystallography and NMR. Yale University Press, New Haven, Conn.) and were further optimized in Refmac (Murshudov, G. N.; Vagin, A. A., and Dodson, E. I. Acta Cryst. 1997, D53, 240).Crystallographic Data and Refinement StatisticsPDB accession code 3O2XData collection statisticsRadiation sourceRadiation detectorSpace groupResolution (Å)Observed reflectionsUnique reflectionsCompleteness (%)Mean I/σI Rsym%bRefinement statisticsResolution (Å)No. protein + ligand atomsNo. solvent atomsR (%), Rfree (%)Wilson B (Å2), Refined <B> (Å2)     Rmsd ideal bond lengths (Å)     Bond angles (°)Rotating anodeMAR CCD 165P21212120.0-1.9 (1.97-1.90)271,72356,98084.3, 79.316.4, 3.88.4, 27.120-1.95,48668217.9, 21.112.9,0.0061.095a Highest resolution bin. b Rsym = Σ(|Ii-<I>|)/ΣIi.  All reflections with I/σI < -1.0 eliminated from scaling. 

MMP-13 Selective α-sulfone Hydroxamates: a Survey of P1\u27 Heterocyclic Amide Isosteres

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